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Ion regulation at gills precedes gas exchange and the origin of vertebrates

Nature volume 610, pages 699–703 (2022)Cite this article

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Abstract

Gas exchange and ion regulation at gills have key roles in the evolution of vertebrates1,2,3,4. Gills are hypothesized to have first acquired these important homeostatic functions from the skin in stem vertebrates, facilitating the evolution of larger, more-active modes of life2,3,5. However, this hypothesis lacks functional support in relevant taxa. Here we characterize the function of gills and skin in a vertebrate (lamprey ammocoete; Entosphenus tridentatus), a cephalochordate (amphioxus; Branchiostoma floridae) and a hemichordate (acorn worm; Saccoglossus kowalevskii) with the presumed burrowing, filter-feeding traits of vertebrate ancestors6,7,8,9. We provide functional support for a vertebrate origin of gas exchange at the gills with increasing body size and activity, as direct measurements in vivo reveal that gills are the dominant site of gas exchange only in ammocoetes, and only with increasing body size or challenges to oxygen supply and demand. Conversely, gills of all three taxa are implicated in ion regulation. Ammocoete gills are responsible for all ion flux at all body sizes, whereas molecular markers for ion regulation are higher in the gills than in the skin of amphioxus and acorn worms. This suggests that ion regulation at gills has an earlier origin than gas exchange that is unrelated to vertebrate size and activity—perhaps at the very inception of pharyngeal pores in stem deuterostomes.

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Fig. 1: Gill contributions to gas exchange and ion regulation in ammocoetes (E. tridentatus).
Fig. 2: Gas exchange in whole and halved acorn worms (S. kowalevskii).
Fig. 3: Ionocyte marker activity and expression in amphioxus (B. floridae) and acorn worms (S. kowalevskii).
Fig. 4: Putative ionocytes in the pharyngeal gill bars of amphioxus (B. floridae) and acorn worms (S. kowalevskii).

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Data availability

All data generated and analysed by this study are publicly accessible (https://doi.org/10.5281/zenodo.7022487). NCBI accession numbers for species and sequences used in gene expression are as follows: txid7739 (M97571.1, XM_002605269.1, XM_002594257.1, XM_002604676.1, XM_002608239.1 and XM_002597600.1) and txid10224 (L28054.1, XM_002736626.2, XM_006814054.1, NM_001184809.1, XM_002734648.1 and XM_002741628.2). NCBI accession numbers for species and sequences used in protein alignments are as follows: txid7739 (XP_002605315.1, XP_002594302.1, XP_002608285.1 and XP_002597646.1), txid10224 (XP_002736672.2, XP_006814117.1, XP_002734694.1 and XP_002741674.2), txid7955 (NP_001161738.1, NP_001032314.1, NP_001289693.1, NP_001032198.1, XP_696967.3, XP_009296364.1, NP_001315073.1, NP_001075158.1, NP_954685.2, NP_001107879.2, NP_001159683.1, NP_001104671.1, XP_009295179.1, NP_957107.1, NP_001017571.2, XP_694982.3, NP_571142.1, NP_571577.1, NP_859424.1, NP_957278.1, NP_944598.2, NP_944599.2, NP_944600.1, NP_001070174.2, NP_956196.1, NP_001030156.1, XP_021335149.1, XP_021334721.1, NP_001106952.1, NP_001107567.1, XP_00929331.1, NP_001106943.1, NP_001091726.2, NP_001025248.2 and NP_001008586.1) and txid9606 (NP_000333.1, NP_001186621.1, NP_005061.3, NP_001076002.1, NP_001209.1, NP_940986.1, NP_036245.1, NP_000058.1, NP_000708.1, NP_001354154.1, NP_001257431.1, NP_001014435.1, NP_001308767.1, NP_001207.2, NP_005240.3, NP_003914.1, NP_005241.1, NP_036320.2, NP_658982.1, NP_997309.2, NP_001129121.1, NP_001445.2, NP_001032242.1, NP_001091954.1, NP_597812.1, NP_001171486.1, NP_003038, NP_003039, NP_004165, NP_001310902.1, NP_001171122.1, NP_001244220.1 and NP_001247420.1). Relevant accession numbers are also provided in the figure source data files. A putative DNA-binding domain was identified in NP_005241.1 using the PROSITE database (https://prosite.expasy.org). Source data are provided with this paper.

Change history

  • 28 October 2022

    In the PDF version of this article initially published, Extended Data Figure 5 was an incorrect image and has now been replaced in the PDF version of the article. The HTML version was unaffected.

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Acknowledgements

We thank P. J. Rombough for discussion that helped inspire this work. This study was funded in part by Natural Sciences and Engineering Council of Canada Discovery Grants to C.J.B. (2018-04172) and C.B.C. (1283784), and a Royal Society University Research Fellowship (UF130182, URF\R\191007) and Royal Society Research Fellows Enhancement Award (RGF/EA/180087) to J.A.G. M.A.S. was supported by an NSERC CGS-D scholarship.

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Authors and Affiliations

  1. Department of Zoology, University of British Columbia, Vancouver, British Columbia, Canada

    Michael A. Sackville & Colin J. Brauner

  2. Département de Sciences Biologiques, Université de Montréal, Montréal, Quebec, Canada

    Christopher B. Cameron

  3. Department of Zoology, University of Cambridge, Cambridge, UK

    J. Andrew Gillis

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  1. Michael A. Sackville
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  2. Christopher B. Cameron
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Contributions

M.A.S., C.J.B. and C.B.C. conceived the study. M.A.S. performed all experiments and data analyses and wrote the manuscript. J.A.G. oversaw all microscopy. All authors provided manuscript edits and comments and approved the final version.

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Correspondence to Michael A. Sackville.

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Extended data figures and tables

Extended Data Fig. 1 Cutaneous diffusive capacity of ammocoetes (E. tridentatus).

Mass-specific surface area (a; n = 15) and epidermal thickness (b; n = 16) measured in different subsets of ammocoetes. Diffusive capacity of the skin (c) calculated from allometric curves fitted to epidermal thickness (a) and mass-specific surface area (b). All data expressed as a function of wet mass (g).

Source data

Extended Data Fig. 2 Gill contributions to CO2 and calcium flux in ammocoetes (E. tridentatus).

Gill contributions (a) and whole-body flux rates (b, c) for CO2 excretion (white), oxygen uptake (grey) and calcium uptake (green) in normoxia at 20 °C. Individual data points plotted as a function of wet mass (g), n = 8 for all groups except n = 7 for gill contributions to calcium uptake

Source data

Extended Data Fig. 3 Whole-body rates of gas and ion flux in ammocoetes (E. tridentatus).

Rates of oxygen uptake (a; n = 40), ammonia excretion (b; n = 36) and sodium uptake (c; n = 21) in normoxia at 10 °C, 26 °C (grey; oxygen only, n = 26) or hypoxia at 20 °C (white; oxygen only, n = 22). Individual data points plotted as a function of wet mass (g)

Source data

Extended Data Fig. 4 ATPase activities in acorn worms (S. kowalevskii), amphioxus (B. floridae) and ammocoetes (E. tridentatus).

Na+/K+-ATPase (a–c; NKA) and V-H+-ATPase (d,e; VHA) activities in multiple tissues of S. kowalevskii (a,d), B. floridae (b,e) and E. tridentatus (c). Data presented as means±sd with individuals superimposed (n = 10 for all tissues except n = 9 for proboscis and hepatic caecum). One-way ANOVA with Tukey’s test (a,b,d) or Kruskal-Wallis with Dunn’s test (c,e), P < 0.05. Letters indicate significant differences between tissues. P < 0.0001 (a–c,e), P = 0.6203 (d).

Source data

Extended Data Fig. 5 Gene expression for ionocyte markers in amphioxus (B. floridae).

Anion exchanger (a; AE, P < 0.0001), sodium–proton exchanger (b; NHE, P < 0.0001), carbonic anhydrase (c; CA, P = 0.0004) and forkhead box protein I (d; FoxI, P < 0.0001) expression in skin, gill, hepatic caecum and muscle of B. floridae. Expression is relative to geometric mean of EF1A and 18S. Data presented as means±sd with individuals superimposed (n = 10). One-way ANOVA with Tukey’s test, P < 0.05. Letters indicate significant differences between tissues

Source data

Extended Data Fig. 6 Gene expression for ionocyte markers in acorn worms (S. kowalevskii).

Anion exchanger (a; AE, P = 0.0163), sodium-proton exchanger (b; NHE, P < 0.0001), carbonic anhydrase (c; CA, P = 0.0431) and forkhead box protein I (d; FoxI, P < 0.0001) expression in skin, gill, intestine and proboscis of S. kowalevskii. Expression is relative to geometric mean of EF1A and 18S. Data presented as means±sd with individuals superimposed (n = 10). Letters indicate significant differences between tissues. One-way ANOVA with Tukey’s test (b,d) or Kruskal-Wallis with Dunn’s test (a,c), P < 0.05

Source data

Extended Data Fig. 7 Origins of gill function.

Our findings support a novel stem chordate or deuterostome origin for ion regulation at gills (pink), perhaps near the inception of pharyngeal gill pores and their role in filter-feeding (black). A vertebrate origin for gas exchange at gills (blue) with increasing body size and activity is also supported by this work, and consistent with fossil and developmental studies (references in text). Data not collected for clades in grey.

Extended Data Fig. 8 Acidic mucins in the foxI+ domains of the pharyngeal gill bars in B. floridae.

Positive staining for Alcian blue shows acidic mucins in the foxI+ domains of the pharyngeal gill bars in B. floridae (outlined section). Counterstained with Nuclear Fast Red, scale bar = 50 μm. This experiment was repeated independently three times with similar results.

Extended Data Table 1 NCBI accession numbers for target proteins and genes in Danio rerio and their reciprocal best BLAST hits in Branchiostoma floridae and Saccoglossus kowalevskii
Full size table
Extended Data Table 2 Primer sequences for qRT–PCR in Branchiostoma floridae (bf) and Saccoglossus kowalevskii (sk)
Full size table

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Sackville, M.A., Cameron, C.B., Gillis, J.A. et al. Ion regulation at gills precedes gas exchange and the origin of vertebrates. Nature 610, 699–703 (2022). https://doi.org/10.1038/s41586-022-05331-7

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